The long-term goal of this research is to understand the genetic changes in the human genome that underlie the evolution of human-specific features of cortical development and to identify their functional consequences. The cerebral cortex of the brain, particularly the prefrontal cortex, has greatly expanded in humans, and this expansion contributes to uniquely human cognitive abilities. Ultimately, an understanding of the molecular basis of human cortical development and function will provide therapeutic targets for diseases such as drug addiction that lead to pathologies in the prefrontal cortex and impaired cognitive function. We recently identified two non-coding RNA genes (HAR1F and HAR1R) encoded by the region of the genome that changed most dramatically in the human lineage since our common ancestor with chimpanzees. These human-specific changes appear to have altered the structure of the HAR1 ncRNAs. Human HAR1F is expressed in a pattern suggesting a role in cortical neuron development. This proposal will explore the function of human and mouse HAR1 ncRNAs through the following specific aims: Aim 1: Characterize the HAR1 expression pattern during neuronal differentiation and determine the biogenesis and subcellular localization of HAR1 transcripts. In order to study the biochemical functions of HAR1 we need to have a source of HAR1 expressing cells. We will determine the expression pattern of HAR1 transcripts in mouse and human embryonic stem cell cultures during neural induction protocols. These cultures will be used to identify the biogenesis pathway, subcellular location and mature form of HAR1 RNAs in the HAR1 expressing cell cultures, which will help us determine whether HAR1 acts to regulate transcription, RNA processing, translation or protein activity. Aim 2: Determine if gain and loss of HAR1 expression leads to changes in neural development in vitro and in vivo. To explore the functional role of HAR1, we will identify pathways and processes altered by modulating HAR1 expression using HAR1-expressing cell cultures and mice. We will examine the downstream effects of modulating HAR1 expression on neural differentiation assays and gene expression by overexpressing HAR1 ncRNAs and by inhibiting endogenous HAR1 expression by RNAi in cultured cells. We will then determine if these perturbations affect neural differentiation in vitro and if they affect gene expression using a microarray based approach. We will also test for cross-regulation between the HAR1F and HAR1R transcripts. Based on the expression pattern of human Har1, we predict that it is involved in cortical neurogenesis or cortical migration. We plan to examine the effect of ectopically expressing HAR1 in differentiating cortical progenitors in vivo using in utero electroporation techniques and test if there are changes in cortical size, patterning, and structure to more fully characterize the role of HAR1 ncRNAs in cortical development. PUBLIC HEALTH RELEVANCE: This project aims to characterize two overlapping non-protein-coding RNA genes we recently identified that are implicated in regulating the development of the human cerebral cortex. These genes are transcribed from the region of the genome most significantly changed in humans since our common ancestor with chimpanzees and may therefore contribute to human-specific aspects of cortical development. A better understanding of human brain development may lead to new approaches for treating neurological disorders associated with cortical pathologies and impaired cognitive functions such as drug abuse.